PROJECT SUMMARY Properly timed gene expression is critical to every aspect of human life. This process is governed by networks of transcription factors that modulate gene expression by binding specific regions in DNA. Yet even today, we are still unraveling the fundamental mechanisms by which a transcription factor identifies a single gene from within the roughly 6 billion base pairs in the genome and then activates it at the right time to regulate cell phenotype. This is especially relevant for cell differentiation, where cell identity is determined by the coordinated expression of hundreds of co-regulated genes that together reprogram undifferentiated stem cells into lineage- specific cells that make up our organs and tissues. Because these changes occur beyond the resolution of conventional light microscopes, most studies rely on biochemical assays to detect protein-DNA binding and chromatin conformation. However, these assays must indirectly infer molecular interactions from population- averaged snapshots using destructive protocols. I propose that we are missing key insights about the molecular dynamics and organizational principles that regulate gene expression. Single molecule fluorescence imaging has emerged as a powerful tool to address these questions. However, our ability to image single molecules in living cells using conventional microscopes is limited by photobleaching and phototoxicity. As such, it remains difficult to link single molecule kinetics obtained from short live-cell movies to emergent cell phenotypes that occur over multiple days. This proposal will overcome this challenge through the development of cutting edge microscopy, microfluidic cell cultures, and genetic models to monitor single molecule dynamics longitudinally in differentiating cells. It will develop a new lattice light sheet microscope to dramatically reduce photobleaching and phototoxicity compared to conventional microscopes. This new instrument will be designed from the ground up around adaptive optical components and custom-made microfluidic devices. Using adipocyte differentiation as a model system and CRISPR-Cas9 gene editing, I will directly image key lineage-determining transcription factors during differentiation. Single molecule trajectories will be used to identify key epigenetic regulatory elements in live cells. By coupling this data to novel computational models, and live-cell reporters of adipogenic gene transcription, we will open a new window into how transcription factor-DNA binding controls gene expression during differentiation. This innovative proposal will develop new technologies and methodologies to image the central dogma with single molecule sensitivity in living cells. These studies will establish a foundation of knowledge for how gene expression is regulated in both normal and disease settings.